The objective of this proposal is the development of a single-cell bioassay for specific proteolytic enzymes associated with neoplasia. The approach involves synthesis of fluorogenic protease substrates that are rendered nondiffusable by covalent attachment of the chromophore to biologically inert supports. Preparation of this new class of spectroscopic probes will be pursued in four stages: 1) synthesis of peptidyl protease substrates that become intensely fluorescent after enzymatic cleavage, 2) measurement of proteolysis kinetics in solution by purified enzymes such as plasminogen activator, plasmin, urokinase, and elastase to determine amino acid sequences that confer substrate selectivity, 3) covalent immobilization of substrates to insoluble, polymeric supports, including polyacrylamide, polystyrene, and glass coverslips, and 4) investigation of these chromogenic probes for their ability to reveal individual cells in population that secrete proteases associated with neoplasia, and to indicate specific regions in tumors that are producing proteolytic activity.